Method and reagent for inhibiting herpes simplex virus replication

ABSTRACT

An enzymatic RNA molecule which specifically cleaves a herpes simplex virus mRNA molecule

This Application is a continuation of U.S. Ser. No. 08/835,269, filedApr. 8, 1997 now U.S. Pat. No. 5,972,699, which is a continuation ofU.S. Ser. No. 08/623,891 filed Mar. 25, 1996 now U.S. Pat. No.5,795,778, which is a continuation of Ser. No. 08/238,200 now abandoned,filed May 4, 1994, which is a continuation of Ser. No. 07/987,133, filedon Dec. 7, 1992 now abandoned, which is a continuation-in-part of U.S.Ser. No. 07/948,359, filed Sep. 18, 1992, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 07/882,921, now abandoned, filedMay 14, 1992, the entirety of each of these applications, including thedrawings, are hereby incorporated by reference herein.

SUMMARY OF THE INVENTION

This invention relates to reagents useful as inhibitors of herpessimplex virus (HSV) replication and gene expression.

The following is a discussion of relevant art, none of which is admittedto be prior art to the pending claims.

Human herpesviruses cause a wide variety of diseases which result insignificant levels of morbidity and mortality worldwide. The HSV groupaccounts for about one million new cases of infection each year in theUnited States. These infections are maintained for the lifetime of thepatient as latent viral infections, which can be stimulated toreactivate by a variety of factors. The manifestations of HSV infectionrange from mild infections of herpes labialis to more serious infectionssuch as herpes encephalitis.

HSV contains a double-stranded DNA genome within its central core, has amolecular weight of approximately 100 million, and a genome encoding atleast 70 polypeptides. The DNA core is surrounded by a capsidconstructed from capsomers arranged in icosapentahedral symmetry.Tightly adherent to the capsid is the tegument, which appears to consistof amorphous material. Loosely surrounding the capsid and tegument is alipid bilayer envelope containing polyamines, lipids, and the viralglycoproteins. These glycoproteins confer distinctive properties to thevirus and provide unique antigens to which the host is capable ofresponding. Glycoprotein G (gG), for example, confers antigenicspecificity to HSV, and therefore results in an antibody response thatcan be used to distinguish HSV-1 (gG-1) from HSV-2 (gG-2).

Replication of HSV is a multi-step process. Following the onset ofinfection, DNA is uncoated and transported to the nucleus of the hostcell. Transcription of immediate-early genes encoding various regulatoryproteins follows. Expression of immediate-early gene products is thenfollowed by the expression of proteins encoded by early and then lategenes, including structural proteins as well as proteins necessary forviral replication. Assembly of the viral core and capsid takes placewithin the nucleus. This is followed by envelopment at the nuclearmembrane and transport out of the nucleus through the endoplasmicreticulum and the Golgi apparatus, where viral envelope proteins areglycosylated. Mature virons are transported to the outer membrane of thehost cell, and release of progeny virus is accompanied by cell death.Replication for all herpesviruses is considered inefficient, with a highratio of noninfectious to infectious viral particles.

The complete sequence of the HSV-1 genome is known. McGeoch et al., 69J. Gen. Virol. 1531, 1988; McGeoch et al., 14 Nucleic Acid Res. 1727,1986; and the elucidation of the HSV-2 genome sequence is underway inlaboratories worldwide. The two subtypes of HSV, HSV-1 and HSV-2, are60-80% homologous at the DNA level, but intragenic variation, whereknown, is less.

Antiviral drugs including acyclovir have been used to effectively treatHSV infections, although with limited success. For example, chronictreatment with acyclovir has resulted in the development ofacyclovir-resistant strains. Nucleoside analogs, such as acycloguanosineand tri-fluorothymidine are currently used for treatment of mucosal andocular HSV infections, but these compounds have little if any effectupon recurrent or secondary infections (which are becoming moreprevalent as the number of HIV-immunosuppressed patients rises). Inaddition, nucleoside analogs are poorly soluble in aqueous solutions,are rapidly catabolized intracellularly, and can be extremely toxic.

SUMMARY OF THE INVENTION

The invention features novel enzymatic RNA molecules, or ribozymes, andmethods for their use for inhibiting HSV replication. Such ribozymes canbe used in a method for treatment of diseases caused by these viruses inman and other animals, including other primates.

Ribozymes are RNA molecules having an enzymatic activity which is ableto repeatedly cleave other separate RNA molecules in a nucleotide basesequence specific manner. Such enzymatic RNA molecules can be targetedto virtually any RNA transcript, and efficient cleavage achieved invitro. Kim et al., 84 Proc. Natl. Acad. of Sci. USA 8788, 1987, Haseloffand Gerlach, 334 Nature 585, 1988, Cech, 260 JAMA 3030, 1988, andJefferies et al., 17 Nucleic Acid Research 1371, 1989.

Ribozymes act by first binding to a target RNA. Such binding occursthrough the target RNA binding portion of a ribozyme which is held inclose proximity to an enzymatic portion of the RNA which acts to cleavethe target RNA. Thus, the ribozyme first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After a ribozyme has bound and cleavedits RNA target it is released from that RNA to search for another targetand can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over othertechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the effective concentration of ribozyme necessary to effect atherapeutic treatment is lower than that of an antisenseoligonucleotide. This advantage reflects the ability of the ribozyme toact enzymatically. Thus, a single ribozyme molecule is able to cleavemany molecules of target RNA. In addition, the ribozyme is a highlyspecific inhibitor, with the specificity of inhibition depending notonly on the base pairing mechanism of binding, but also on the mechanismby which the molecule inhibits the expression of the RNA to which itbinds. That is, the inhibition is caused by cleavage of the RNA targetand so specificity is defined as the ratio of the rate of cleavage ofthe targeted RNA over the rate of cleavage of non-targeted RNA. Thiscleavage mechanism is dependent upon factors additional to thoseinvolved in base pairing. Thus, it is thought that the specificity ofaction of a ribozyme is greater than that of antisense oligonucleotidebinding the same RNA site.

These ribozymes exhibit a high degree of specificity for only thevirally encoded mRNA in infected cells. Ribozyme molecules targeted tohighly conserved sequence regions will allow the treatment of manyspecies or subtypes of HSV with a single compound. There is noacceptable treatment which will give a broad spectrum of activity withno toxic side effects. No treatment exists which specifically attacksviral gene expression which is responsible for the transformation ofepithelial cells by HSV, for the maintenance of the episomal genome inlatently infected cells or for the vegetative replication of the virusin permissive cells.

The methods of this invention can be used to treat HSV infections, whichincludes these diseases noted above. The utility can be extended toother HSV-like virus which infect non-human primates where suchinfections are of veterinary importance.

Thus, in the first aspect the invention features an enzymatic RNAmolecule (or ribozyme) which specifically cleaves HSV expressed RNA. Theribozymes of the invention are capable of specifically cleavingparticular viral mRNA targets, resulting in the destruction of mRNAtranscript integrity required for translation, and therefore preventingthe synthesis of the encoded protein. More specifically, the ribozymesof the invention are targeted to and prevent the translation of mRNAsencoding proteins required for viral genomic replication, virionstructure, and viral infectivity, maintenance of the latent state, etc.,and therefore interfere with critical events required for viralsurvival. Thus, diseases caused by HSV may be effectively treated byribozyme-mediated interruption of the viral life-cycle.

Preferred cleavage sites are at genes required for viral replication,e.g., protein synthesis, such as in the immediate early genes (ICP0,ICP4, ICP22 and ICP27), genes required for nucleic acid metabolism(UL13, 39, 40, 50), host shut-off (UL41), control of late viral proteinsynthesis (γ 34.5), DNA replication (UL5, 8, 9, 29, 30, 42, 53) andstructural protein encoding genes (gB and gC).

Alternative regions make suitable targets of ribozyme-mediatedinhibition of HSV replication. Most preferred targets include ICP4(IE3),ICP27(UL54), UL39, UL40, UL5, γ 34.5 and UL27(gB) genes. Below areprovided 5 examples of ribozymes targeted to the ICP4 gene but othersuch ribozymes are expected to have utility at these other genes.

By “enzymatic RNA molecule” it is meant an RNA molecule which hascomplementarity in a substrate binding region to a specified genetarget, and also has an enzymatic activity which is active tospecifically cleave RNA in that target. That is, the enzymatic RNAmolecule is able to intermolecularly cleave RNA and thereby inactivate atarget RNA molecule. This complementarity functions to allow sufficienthybridization of the enzymatic RNA molecule to the target RNA to allowthe cleavage to occur. 100% complementarity is preferred, butcomplementarity as low as 50-75% may also be useful in this invention.By “equivalent” RNA to HSV is meant to include those naturally occurringviral encoded RNA molecules associated with viral caused diseases invarious animals, including humans, and other primates. These viralencoded RNAs have similar structures and equivalent genes to each other.

In preferred embodiments, the enzymatic RNA molecule is formed in ahammerhead motif, but may also be formed in the motif of a hairpin,hepatitis delta virus, group I intron or RNAseP RNA (in association withan RNA guide sequence). Examples of such hammerhead motifs are describedby Rossi et al., 8 Aids Research and Human Retroviruses 183, 1992, ofhairpin motifs by Hampel et al., “RNA Catalyst for Cleaving Specific RNASequences”, filed Sep. 20, 1989, which is a continuation-in-part of U.S.Ser. No. 07/247,100 filed Sep. 20, 1988, Hampel and Tritz, 28Biochemistry 4929, 1989 and Hampel et al., 18 Nucleic Acids Research299, 1990, and an example of the hepatitis delta virus motif isdescribed by Perrotta and Been, 31 Biochemistry 16, 1992, of the RNAsePmotif by Guerrier-Takada et al., 35 Cell 849, 1983, and of the group Iintron by Cech et al., U.S. Pat. No. 4,987,071. These specific I motifsare not limiting in the invention and those skilled in the art willrecognize that all that is important in an enzymatic RNA molecule ofthis invention is that it has a specific substrate binding site which iscomplementary to one or more of the target gene RNA regions, and that ithave nucleotide sequences within or surrounding that substrate bindingsite which impart an RNA cleaving activity to the molecule.

In particularly preferred embodiments, the RNA which is cleaved is HSVICP4 or UL5 mRNA regions (for UL5, nucleotide #1 is defined bypreliminary mapping of the cap site in our laboratory and nucleotide2800 is the site of translation initiation for the UL4 protein whoseopen reading frame is also included in the UL5 mRNA) selected from oneor more of the following sequences:

TABLE I ICP4 nucleotide number sequence 52 AGAGACAGACCGUCAGACGCUC (SEQ.ID. NO. 1) 81 CCGGGACGCCGAUAC (SEQ. ID. NO. 2) 116 GGAUCGGCCGUCCCUGUCCU(SEQ. ID. NO. 3) 143 ACCCAAGCAUCGACCGGUCC (SEQ. ID. NO. 4) 212GGUCUCGCCCCCUCCCCCC (SEQ. ID. NO. 5) 236 UAGGUGACCUACCGUGCUACGUCCGCCGUCG(SEQ. ID. NO. 6) 273 UAUCCCCGGAGGA (SEQ. ID. NO. 7) 304GGCGUCGGAGAACAAGCAGCGCCCCGGCUCC (SEQ. ID. NO. 8) 458 CACGACCUCGAC (SEQ.ID. NO. 9) 532 CGCCGUCUCGCCGCGACAGCUGGCUCUGCUG (SEQ. ID. NO. 10) 584GUCCGGACGAUCCCGACGCCC (SEQ. ID. NO. 11) 647GACGACGAUGACGGGGACGAGUACGACGACG (SEQ. ID. NO. 12) 745 GUAUCCGGACCCCAC(SEQ. ID. NO. 13) 803 CGUCGUCACGGCC (SEQ. ID. NO. 14) 834 CAUAGACCT(SEQ. ID. NO. 15) 871 GUCCGCAUCCUCU (SEQ. ID. NO. 16) 1092 GCAUCGAGCGCC(SEQ. ID. NO. 17) 1140 GGGCCGCUUCACGGCCGGGCAG (SEQ. ID. NO. 18) 1345GCGACGCCGGUUCGAGGC (SEQ. ID. NO. 19) 1419 ACGCCCUGAUCACG (SEQ. ID. NO.20) 1465 GGGGUGGCUCCAGAACC (SEQ. ID. NO. 21) 1547AACAGCAGCUCCUUCAUCACCGGCAGCGUGG (SEQ. ID. NO. 22) 1632 GCCUGGCGCACGC(SEQ. ID. NO. 23) 1652 CCGUGGCCAUGA (SEQ. ID. NO. 24) 1672 AUACGACCGCGC(SEQ. ID. NO. 25) 1682 GCGCAGAAGGGCUUCCUGCUGAC (SEQ. ID. NO. 26) 2012GCCUGCCGCGGGAUCCUGGAGGCGCUGG (SEQ. ID. NO. 27) 2368CCUGCUGUUUGACAACCAGAGCCUGC (SEQ. ID. NO. 28) 2483 AAGCGCAAGAGUCCC (SEQ.ID. NO. 29) 2585 GCCCCCCUCCCCGCGCC (SEQ. ID. NO. 30) 2610 CCUCCACGCCCC(SEQ. ID. NO. 31) 2686 GCGCCCCGUGGCCGUGUCG (SEQ. ID. NO. 32) 2794CCUGGAGGCCUACUGCUCCCCG (SEQ. ID. NO. 33) 2846 CUGUUCCCCGUCCCCUGGCGAC(SEQ. ID. NO. 34) 2874 UCAUGUUUGACCC (SEQ. ID. NO. 35) 2895UGGCCUCGAUCGCCGCGCGGUGCGCC (SEQ. ID. NO. 36) 2960GACGACGACGAUAACCCCCACCC (SEQ. ID. NO. 37) 3165 AUCCCCGACCCCGAGGACGUGCGC(SEQ. ID. NO. 38) 3243 CCCGACGUGUCG (SEQ. ID. NO. 39) 4038GUGCUGGCGGCGGCGGGGGCCGUGGA (SEQ. ID. NO. 40) 4076 GGAGGCGGGCUUGGCCAC(SEQ. ID. NO. 41) 4130 CUGGGACGAAGAC (SEQ. ID. NO. 42) 4168GGGUGCUGUAACGG (SEQ. ID. NO. 43) 1 GUGAACCUUUACCCAGCCGUCCUC (SEQ. ID.NO. 44) 30 GCACAGCGCUUCCGUG (SEQ. ID. NO. 45) 110AGCGCCAGCUAGACGGACAGAAA (SEQ. ID. NO. 46) 145 CACCUUCAGCAACCCGGG (SEQ.ID. NO. 47) 237 UAAGCGCAUCCGA (SEQ. ID. NO. 48) 255 CUCGCAACAAC (SEQ.ID. NO. 49) 278 CGCAAGUGCCCCAUCUGCAGUGGUUCCG (SEQ. ID. NO. 50) 313GCGGCCUUAGAGUCCCCCGC (SEQ. ID. NO. 51) 363 GGUGUAUCUUAUCACCGGCAA (SEQ.ID. NO. 52) 389 GCUCCGGAAAGAGCA (SEQ. ID. NO. 53) 412CAGACAAUCAACGAGGUCUUGGA (SEQ. ID. NO. 54) 440UGGUGACGGGCGCCACGCGCAUUGCGGC (SEQ. ID. NO. 55) 468 CCAAAACAUGUACGCC(SEQ. ID. NO. 56) 515 UCAACACCAUCUUUCAUGAAUU (SEQ. ID. NO. 57) 564CCAACUGGGACAGUACCCGUACACCCUGACCA (SEQ. ID. NO. 58) 617ACCUGCAGCGACGAGAUCUGACGUACUACUGG (SEQ. ID. NO. 59) 667ACGAAGCGCGCCCUGGCCG (SEQ. ID. NO. 60) 765 CCUGACGCGGUUGGCCC (SEQ. ID.NO. 61) 807 CUUUACCCGCAGCAA (SEQ. ID. NO. 62) 827 UCGUCAUCGACGAG (SEQ.ID. NO. 63) 841 GCCGGGCUCCUUGGGCGUCACCUCC (SEQ. ID. NO. 64) 871GCCGUGGUGUAUU (SEQ. ID. NO. 65) 926 CGGCCCGCCUGCGGCC (SEQ. ID. NO. 66)981 CCUGGAGUCGACCUUC (SEQ. ID. NO. 67) 1020 CGUCCGCCAGA (SEQ. ID. NO.68) 1052 UCAUCUGCAACCGCACGCUGCGCGAGUACGCC (SEQ. ID. NO. 69) 1084CGCCUCUCGUAUAGCUGGGCCA (SEQ. ID. NO. 70) 1106 UUUJUAUUAACAACAAAC (SEQ.ID. NO. 71) 1148 ACCUCAUGAAGGUGCUGGAGUACGGCC (SEQ. ID. NO. 72) 1170GGCCUGCCCAUCACCCAGGAGCACAUGC (SEQ. ID. NO. 73) 1212CCGGAAAACUACAUCACCAACC (SEQ. ID. NO. 74) 1234 CCGCCAACCUCCCCGGCUGGA(SEQ. ID. NO. 75) 1271 UGUUCUCCUCCCACAAAGAGGUGAGCGCGU (SEQ. ID. NO. 76)1333 ACCCGUGAGGG (SEQ. ID. NO. 77) 1372 CUUACGUUCGU (SEQ. ID. NO. 78)1389 CAAGGAGUUUGACGAAU (SEQ. ID. NO. 79) 1428 CGGCCUGACGAUUGAAAA (SEQ.ID. NO. 80) 1471 AUCACCAACUACUCGCAGAGCCAGG (SEQ. ID. NO. 81) 1522GAGGUGCACAGCAAACA (SEQ. ID. NO. 82) 1544 UGGUCGUGGCCCGCAAC (SEQ. ID. NO.83) 1576 CUCAACAGCCAGAUCGCGGUGACCGC (SEQ. ID. NO. 84) 1602GCGCCUGCGAAAACUGGUUUUU (SEQ. ID. NO. 85) 1673 GCUUUGUAAAGACUC (SEQ. ID.NO. 86) 1760 ACAACUUUCUGCAGCGCCCG (SEQ. ID. NO. 87) 1788 UGCGACCCAGA(SEQ. ID. NO. 88) 1805 UCGCCUACGCCCGCAUGGGAGAACUAACGG (SEQ. ID. NO. 89)1915 UUGAUUUUAAGCAAC (SEQ. ID. NO. 90) 1959 CCCGGACGAUU (SEQ. ID. NO.91) 2006 UGGACGAAC (SEQ. ID. NO. 92) 2014AACAGCUCGACGUGUUUUACUGCCACUACACC (SEQ. ID. NO. 93) 2068CCGCCGUUCACACCCAGUUUGCGC (SEQ. ID. NO. 94) 2301CGGGCCUUCCUCGGGAGAUUCCGAAU (SEQ. ID. NO. 95) 2132 AAGAGCUCUUCGG (SEQ.ID. NO. 96) 2150 CAUUUGAAGUCGCCCC (SEQ. ID. NO. 97) 2174CGUACGUGGACAACGUUAUCUUCCGGGGCU (SEQ. ID. NO. 98) 2209 AUGCUGACCGG (SEQ.ID. NO. 99) 2225 CGCGCGGGGGGCUGAUGUCCGUC (SEQ. ID. NO. 100) 2255AGACGGACAAUUAUACGCUCAU (SEQ. ID. NO. 101) 2289 CGCACGGGUGUUU (SEQ. ID.NO. 102) 2339 CCAACGUGGCCGAGUUACUGGAAGAGG (SEQ. ID. NO. 103) 2366CCCCCCUGCCU (SEQ. ID. NO. 104) 2398 CACGGCUUCAUGUCCGUCGUCAACAC (SEQ. ID.NO. 105) 2418 CAACACCCAACAUCA (SEQ. ID. NO. 106) 2467GCCAUGGCCAUAAACGCCGACUACGGCAU (SEQ. ID. NO. 107) 2546ACAAGGUCGCCAUCUGCUUUACGCCC (SEQ. ID. NO. 108) 2572 GGCAACCUGCGCCUCAAC(SEQ. ID. NO. 109) 2618 CCUCCUCCGAAUUCCUUCGCAU (SEQ. ID. NO. 110) 2673CGAUGACGUCAU (SEQ. ID. NO. 111) 2701 UCGGCUCUGCGCGAUCCGAACGUGGUCAUUG(SEQ. ID. NO. 112) 2732 UCUAUUAACCCGCCGUCCCCUUAC (SEQ. ID. NO. 113) 2776GGGGGACUCACUACCCACC (SEQ. ID. NO. 114) 2795 GCGAGAUGUCCAAUCCACAGACG(SEQ. ID. NO. 115)

In a second related aspect, the invention features a mammalian cellwhich includes an enzymatic RNA molecule as described above. Preferably,the mammalian cell is a human or other primate cell.

In a third related aspect, the invention features an expression vectorwhich includes nucleic acid encoding the enzymatic RNA moleculesdescribed above, located in the vector, e.g., in a manner which allowsexpression of that enzymatic RNA molecule within a mammalian cell.

In a fourth related aspect, the invention features a method fortreatment of a HSV-caused disease by administering to a patient anenzymatic RNA molecule which cleaves HSV-encoded RNA or related RNA inthe regions discussed above.

The invention provides a class of chemical cleaving agents which exhibita high degree of specificity for the viral RNA of HSV-infected cells.The ribozyme molecule is preferably targeted to a highly conservedsequence region of an HSV such that all types and strains of this viruscan be treated with a single ribozyme. Such enzymatic RNA molecules canbe delivered exogenously to infected cells. In the preferred hammerheadmotif the small size (less than 40 nucleotides, preferably between 32and 36 nucleotides in length) of the molecule allows the cost oftreatment to be reduced compared to other ribozyme motifs.

Synthesis of ribozymes greater than 100 nucleotides in length is verydifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. Delivery of ribozymes by expression vectors isprimarily feasible using only ex vivo treatments. This limits theutility of this approach. In this invention, small ribozyme motifs(e.g., of the hammerhead structure, shown generally in FIG. 1) are usedfor exogenous delivery. The simple structure of these molecules alsoincreases the ability of the ribozyme to invade targeted regions of themRNA structure. Thus, unlike the situation when the hammerhead structureis included within longer transcripts, there are no non-ribozymeflanking sequences to interfere with correct folding of the ribozymestructure or with complementary binding of the ribozyme to the mRNAtarget region.

The enzymatic RNA molecules of this invention can be used to treat HSVinfections. Infected animals can be treated at the time of productiveinfection. This timing of treatment will reduce viral loads in infectedcells and disable viral replication in any subsequent rounds ofinfection. This is possible because the preferred ribozymes disablethose structures required for successful initiation of viral proteinsynthesis. For treatment of transformed cervical epithelia orkeratinocytes, the method of this invention will inhibit the expressionof viral genes known to cause cell immortalization. For treatment oflatent viral infections, this invention will inhibit gene expressionrequired for the maintenance of the viral episomal genome.

The preferred targets of the present invention are advantageous overother targets since they act not only during the productive infectionbut also in latently infected cells and in virally transformed cells. Inaddition, viral particles which are released during a first round ofinfection in the presence of such ribozymes will still be immunogenic byvirtue of having their capsids intact. Thus, one method of thisinvention allows the creation of defective but immunogenic viralparticles, and thus a continued possibility of initiation of an immuneresponse in a treated animal.

In addition, the enzymatic RNA molecules of this invention can be usedin vitro in a cell culture transfected with HSV DNA to produce defectiveviral particles. These particles can then be used for instigation ofimmune responses in a prophylactic manner, or as a treatment of infectedanimals.

Ribozymes of this invention may be used as diagnostic tools to examinegenetic drift and mutations within diseased cells. The closerelationship between ribozyme activity and the structure of the targetRNA allows the detection of mutations in any region of the moleculewhich alters the base-pairing and three-dimensional structure of thetarget RNA. By using multiple ribozymes described in this invention, onemay map nucleotide changes which are important to RNA structure andfunction in vitro, as well as in cells and tissues. Cleavage of targetRNAs with ribozymes may be used to inhibit gene expression and definethe role (essentially) of specified gene products in the progression ofdisease. In this manner, other genetic targets may be defined asimportant mediators of the disease. These experiments will lead tobetter treatment of the disease progression by affording the possibilityof combinational therapies (e.g., multiple ribozymes targeted todifferent genes, ribozymes coupled with known small molecule inhibitors,or intermittent treatment with combinations of ribozymes and/or otherchemical or biological molecules). (See, Thompson and Draper, “Methodand Reagent for Treatment of Neuroblastoma”; Draper, “Method and Reagentfor Treatment of a Stenotic Condition; Sullivan and Draper, “Method andReagent for Treatment of Inflammatory Disease”; and Draper, “Method andReagent for Treatment of Arthritic Conditions”; all filed on the samedate as the present application, and all hereby incorporated byreference herein.)

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTIONS OF THE PREFERRED EMBODIMENTS

The drawing will first briefly be described.

DRAWING

FIG. 1 is a diagrammatic representation of a hammerhead motif ribozymeshowing stems I, II and III (marked (I), (II) and (III) respectively)interacting with a target region. The 5′ and 3′ ends of both ribozymeand target are shown. Dashes indicate base-paired nucleotides.

TARGET SITES

Those regions (genes) of the genome which are essential for HSVreplication are expected to maintain a constant sequence (i.e., areconserved) over extensive periods of time. These regions are preferredtarget sites in this invention since they are more likely to beconserved between different types or strains of HSV, and thus only oneribozyme is needed to destroy all viral RNA. Thus, one ribozyme may beused to target all HSV-encoded RNA. We have selected several such genesof these viruses, and analyzed the structure of the encoded mRNAs usingRNA-fold computer analyses. We have identified open structures in theRNAs which may be cleaved by ribozymes targeted to those regions. Tworegions analyzed in detail are the HSV-1 ICP4 and UL5 mRNAs; other genescan be analyzed in a manner similar to that described below.

Ribozymes targeting selected regions of the HSV RNA are preferablychosen to cleave the target RNA in a manner which inhibits translationof the RNA. Genes are selected such that such viral replication isinhibited, e.g., by inhibiting protein synthesis. Selection of effectiveribozymes to cleave target sites within these critical regions of viralmRNA entails testing the accessibility of the target mRNA tohybridization with various oligonucleotide probes. These studies can beperformed using RNA probes and assaying accessibility by cleaving thehybrid molecule with RNAseH (see below). Alternatively, such a study canuse ribozyme probes designed from secondary structure predictions of theRNAs, and assaying cleavage products by polyacrylamide gelelectrophoresis (PAGE), to detect the presence of cleaved and uncleavedmolecules.

HSV specific ribozymes may be designed to also inhibit otherherpesviruses, particularly varicella zoster virus (VZV), which exhibitssignificant intragenic nucleotide homology with a number of analogousHSV genes. For example, the VZV immediate-early protein IE62 exhibitsconsiderable amino acid homology to the HSV immediate-early regulatoryprotein ICP4/IE3 and is a major component of VZV particles (Kinchingtonet al., 66 J. Virol. 359, 1992). Also, some transcription units andtheir encoded proteins are conserved within the genomes ofevolutionarily divergent human herpesviruses, and ribozyme targetswithin these genetic units may therefore provide the basis for designingribozymes with broad therapeutic applications.

The following is but one example of a method by which suitable targetsites can be identified and is not limiting in this invention.Generally, the method involves identifying potential cleavage sites fora hammerhead ribozyme, and then testing each of these sites to determinetheir suitability as targets by ensuring that secondary structureformation is minimal.

The mRNA sequences of the viruses are analyzed throughout the regionsdiscussed above (using the nucleotide sequence of the HSV-1 ICP4 gene(EMBL No. HEHSV1G3, nucleotides 1176-5435) and UL5 gene). Putativeribozyme cleavage sites are found to be weak or non-base paired regionsof the virus mRNA. These sites represent the preferred sites forhammerhead or other ribozyme cleavage within these target RNAs.

Short RNA substrates corresponding to each of the gene sites aredesigned. Each substrate is composed of two to three nucleotides at the5′ and 3′ ends that will not base pair with a corresponding ribozymerecognition region. The unpaired regions flank a central region of 12-14nucleotides to which complementary arms in the ribozyme are designed.

The structure of each substrate sequence is predicted using a PC foldcomputer program. Sequences which give a positive free energy of bindingare accepted. Sequences which give a negative free energy are modifiedby trimming one or two bases from each of the ends. If the modifiedsequences are still predicted to have a strong secondary structure, theyare rejected.

After substrates are chosen, ribozymes are designed to each of the RNAsubstrates. Ribozyme folding is also analyzed using PC fold.

Ribozyme molecules are sought which form hammerhead motif stem II (seeFIG. 1) regions and contain flanking arms which are devoid ofintramolecular base pairing. Often the ribozymes are modified bytrimming a base from the ends of the ribozyme, or by introducingadditional base pairs in stem II to achieve the desired fold. Ribozymeswith incorrect folding are rejected. After substrate/ribozyme pairs arefound to contain correct intramolecular structures, the molecules arefolded together to predict intermolecular interactions. A schematicrepresentation of a ribozyme with its coordinate base pairing to itscognate target sequence is shown in FIG. 1.

Using such analyses, predictions of effective target sites in the viralmRNAs, based upon computer generated structural analyses, were obtained(see SEQ ID NOS. 1-115). The target region is listed with the nucleotidenumber (in the HSV-1 ICP4 or UL5 mRNA) noted as base number.

Those targets thought to be useful as ribozyme targets can be tested todetermine accessibility to nucleic acid probes in a ribonuclease H assay(see below). This assay provides a quick test of the use of the targetsite without requiring synthesis of a ribozyme. It can be used to screenfor sites most suited for ribozyme attack.

Synthesis of Ribozymes

Ribozymes useful in this invention can be produced by gene transcriptionas described by Cech, supra, or by chemical synthesis. Chemicalsynthesis of RNA is similar to that for DNA synthesis. The additional2′-OH group in RNA, however, requires a different protecting groupstrategy to deal with selective 3′-5′ internucleotide bond formation,and with RNA susceptibility to degradation in the presence of bases. Therecently developed method of RNA synthesis utilizing thet-butyldimethylsilyl group for the protection of the 2′ hydroxyl is themost reliable method for synthesis of ribozymes. The method reproduciblyyields RNA with the correct 3′-5′ internucleotide linkages, with averagecoupling yields in excess of 99%, and requires only a two-stepdeprotection of the polymer.

A method based on H-phosphonate chemistry exhibits a relatively lowercoupling efficiency than a method based upon the phosphoramiditechemistry. This is a problem for synthesis of DNA as well. A promisingapproach to scale-up of automatic oligonucleotide synthesis has beendescribed recently for the H-phosphonates. A combination of a propercoupling time and additional capping of “failure” sequences gave highyields in the synthesis of oligodeoxynucleotides in scales in the rangeof 14 micromoles with as little as 2 equivalents of a monomer in thecoupling step. Another alternative approach is to use soluble polymericsupports (e.g., polyethylene glycols), instead of the conventional solidsupports. This method can yield short oligonucleotides in hundredmilligram quantities per batch utilizing about 3 equivalents of amonomer in a coupling step.

Various modifications to ribozyme structure can be made to enhance theutility of ribozymes. Such modifications will enhance shelf-life,half-life in vitro, stability, and ease of introduction of suchribozymes to the target site, e.g., to enhance penetration of cellularmembranes, and confer the ability to recognize and bind to targetedcells.

Exogenous delivery of ribozymes benefits from chemical modification ofthe backbone, e.g., by the overall negative charge of the ribozymemolecule being reduced to facilitate diffusion across the cell membrane.The present strategies for reducing the oligonucleotide charge include:modification of internucleotide linkages by ethylphosphonates, use ofphosphoramidites, linking oligonucleotides to positively chargedmolecules, and creating complex packages composed of oligonucleotides,lipids and specific receptors or effectors for targeted cells. Examplesof such modifications include sulfur-containing ribozymes containingphosphorothioates and phosphorodithioates as internucleotide linkages inRNA. Synthesis of such sulfur-modified ribozymes is achieved by use ofthe sulfur-transfer reagent, ³H-1,2-benzenedithiol-3-one 1,1-dioxide.Ribozymes may also contain ribose modified ribonucleotides. Pyrimidineanalogues are prepared from uridine using a procedure employingdiethylamino sulphur trifluoride (DAST) as a starting reagent. Ribozymescan also be either electrostatically or covalently attached to polymericcations for the purpose of reducing charge. The polymer can be attachedto the ribozyme by simply converting the 3′-end to a ribonucleosidedialdehyde which is obtained-by a periodate cleavage of the terminal2′,3′-cis diol system. Depending on the specific requirements fordelivery systems, other possible modifications may include differentlinker arms containing carboxyl, amino or thiol functionalities. Yetfurther examples include use of methylphosphonates and 2′-O-methylriboseand 5′ or 3′ capping or blocking with m₇GpppG or m₃ ^(2,2,7)GpppG.

For example, a kinased ribozyme is contacted with guanosine triphosphateand guanyltransferase to add a m³G cap to the ribozyme. After suchsynthesis, the ribozyme can be gel purified using standard procedure. Toensure that the ribozyme has the desired activity, it may be tested withand without the 5′ cap using standard procedures to assay both itsenzymatic activity and its stability.

Synthetic ribozymes, including those containing various modifiers, canbe purified by high pressure liquid chromatography (HPLC). Other liquidchromatography techniques, employing reverse phase columns and anionexchangers on silica and polymeric supports may also be used.

There follows an example of the synthesis of one ribozyme. A solid phasephosphoramidite chemistry is employed. Monomers used are2′-tert-butyl-dimethylsilyl cyanoethylphosphoramidities of uridine,N-benzoyl-cytosine, N-phenoxyacetyl adenosine and guanosine (GlenResearch, Sterling, Va.). Solid phase synthesis is carried out on eitheran ABI 394 or 380B DNA/RNA synthesizer using the standard protocolprovided with each machine. The only exception is that the coupling stepis increased from 10 to 12 minutes. The phosphoramidite concentration is0.1 M. Synthesis is done on a 1 μmole scale using a 1 μmole RNA reactioncolumn (Glen Research). The average coupling efficiencies are between97% and 98% for the 394 model, and between 97% and 99% for the 380Bmodel, as determined by a calorimetric measurement of the releasedtrityl cation.

Blocked ribozymes are cleaved from the solid support (e.g., CPG), andthe bases and diphosphoester moiety deprotected in a sterile vial by dryethanolic ammonia (2 mL) at 55° C. for 16 hours. The reaction mixture iscooled on dry ice. Later, the cold liquid is transferred into a sterilescrew cap vial and lyophilized.

To remove the 2′-tert-butyl-dimethylsilyl groups from the ribozyme, theresidue is suspended in 1 M tetra-n-butylammonium fluoride in dry THF(TBAF), using a 20 fold excess of the reagent for every silyl group, for16 hours at ambient temperature (about 15-25° C). The reaction isquenched by adding an equal volume of sterile 1 M triethylamine acetate,pH 6.5. The sample is cooled and concentrated on a SpeedVac to half theinitial volume.

The ribozymes are purified in two steps by HPLC on a C4 300 Å 5 mmDeltaPak column in an acetonitrile gradient.

The first step, or “trityl on” step, is a separation of 5′-DMT-protectedribozyme(s) from failure sequences lacking a 5′-DMT group. Solvents usedfor this step are: A (0.1 M triethylammonium acetate, pH 6.8) and B(acetonitrile). The elution profile is: 20% B for 10 minutes, followedby a linear gradient of 20% B to 50% B over 50 minutes, 50% B for 10minutes, a linear gradient of 50% B to 100% B over 10 minutes, and alinear gradient of 100% B to 0% B over 10 minutes.

The second step is a purification of a completely deblocked ribozyme bya treatment of 2% trifluoroacetic acid on a C4 300 Å 5 mm DeltaPakcolumn in an acetonitrile gradient. Solvents used for this second stepare: A (0.1 M Triethylammonium acetate, pH 6.8) and B (80% acetonitrile,0.1 M triethylammonium acetate, pH 6.8). The elution profile is: 5% Bfor 5 minutes, a linear gradient of 5% B to 15% B over 60 minutes, 15% Bfor 10 minutes, and a linear gradient of 15% B to 0% B over 10 minutes.

The fraction containing ribozyme is cooled and, lyophilized on aSpeedVac. Solid residue is dissolved in a minimum amount of ethanol andsodium perchlorate in acetone. The ribozyme is collected bycentrifugation, washed three times with acetone, and lyophilized.

Expression Vector

While synthetic ribozymes are preferred in this invention, thoseproduced by expression vectors can also be used. In designing a suitableribozyme expression vector the following factors are important toconsider. The final ribozyme must be kept as small as possible tominimize unwanted secondary structure within the ribozyme. A promoter(e.g., the human cytomegalovirus immediate early region promoter orkeratin promoters from human keratin genes) should be chosen to be arelatively strong promoter, and expressible both in vitro and in vivo.Such a promoter should express the ribozyme at a level suitable toeffect production of enough ribozyme to destroy a target RNA, but not attoo high a level to prevent other cellular activities from occurring(unless cell death itself is desired).

A hairpin at the 5′ end of the ribozyme is useful to ensure that therequired transcription initiation sequence (GG or GGG or GGGAG) does notbind to some other part of the ribozyme and thus affect regulation ofthe transcription process. The 5′ hairpin is also useful to protect theribozyme from 5′-3′ exonucleases. A selected hairpin at the 3′ end ofthe ribozyme is useful since it acts as both a transcription terminationsignal, and as a protection from 3′-5′ exonucleases. One example of aknown termination signal is that present on the T7 RNA polymerasesystem. This signal is about 30 nucleotides in length. Other 3′ hairpinsof shorter length can be used to provide good termination and RNAstability. Such hairpins can be inserted within the vector sequences toallow standard ribozymes to be placed in an appropriate orientation andexpressed with such sequences attached.

Poly(A) tails are also useful to protect the 3′ end of the ribozyme.These can be provided by either including a poly(A) signal site in theexpression vector (to signal a cell to add the poly(A) tail in vivo), orby introducing a poly(A) sequence directly into the expression vector.In the first approach the signal must be located to prevent unwantedsecondary structure formation with other parts of the ribozyme. In thesecond approach, the poly(A) stretch may reduce in size over time whenexpressed in vivo, and thus the vector may need to be checked over time.Care must be taken in addition of a poly(A) tail which binds poly(A)binding proteins which prevent the ribozyme from acting.

Ribozyme Testing

Once the desired ribozymes are selected, synthesized and purified, theyare tested in kinetic and other experiments to determine their utility.An example of such a procedure is provided below.

Preparation of Ribozyme

Crude synthetic ribozyme (typically 350 μg at a time) is purified byseparation on a 15% denaturing polyacrylamide gel (0.75 mm thick, 40 cmlong) and visualized by UV shadowing. Once excised, gel slicescontaining full length ribozyme are soaked in 5 ml gel elution buffer(0.5 M NH₄OAc, 1 mM EDTA) overnight with shaking at 4° C. The eluent isdesalted over a C-18 matrix (Sep-Pak cartridges, Millipore, Milford,Mass.) and vacuum dried. The dried RNA is resuspended in 50-100 μl TE(TRIS 10 mM, EDTA 1 mM, pH 7.2). An aliquot of this solution is diluted100 fold into 1 ml TE, half of which is used to spectrophotometricallyquantitate the ribozyme solution. The concentration of this dilute stockis typically 150-800 nM. Purity of the ribozyme is confirmed by thepresence of a single band on a denaturing polyacrylamide gel.

A ribozyme may advantageously be synthesized in two or more portions.Each portion of a ribozyme will generally have only limited or noenzymatic activity, and the activity will increase substantially (by atleast 5-10 fold) when all portions are ligated (or otherwise juxtaposed)together. A specific example of hammerhead ribozyme synthesis isprovided below.

The method involves synthesis of two (or more) shorter “half” ribozymesand ligation of them together using T4 RNA ligase. For example, to makea 34 mer ribozyme, two 17 mers are synthesized, one is phosphorylated,and both are gel purified. These purified 17 mers are then annealed to aDNA splint strand complementary to the two 17 mers. This DNA splint hasa sequence designed to locate the two 17 mer portions with one end ofeach adjacent each other. The juxtaposed RNA molecules are then treatedwith T4 RNA ligase in the presence of ATP. The 34 mer RNA so formed isthen HPLC purified.

Preparation of Substrates

Approximately 10-30 pmoles of unpurified substrate is radioactively 5′end-labelled with T4 polynucleotide kinase using 25 pmoles of [γ-³²p]ATP. The entire labelling mix is separated on a 20% denaturingpolyacrylamide gel and visualized by autoradiography. The full lengthband is excised and soaked overnight at 4° C. in 100 μl of TE (10 mMTris-HCl pH 7.6, 0.1 mM EDTA).

Kinetic Reactions

For reactions using short substrates (between 8 and 16 bases) asubstrate solution is made 1× in assay buffer (75 mM Tris-HCl, pH 7.6;0.1 mM EDTA, 10 M MgCl₂) such that the concentration of substrate isless than 1 nM. A ribozyme solution (typically 20 nM) is made 1X× inassay buffer and four dilutions are made using 1× assay buffer. Fifteenμl of each ribozyme dilution (i.e., 20, 16, 12, 8 and 4 nM) is placed ina separate tube. These tubes and the substrate tube are pre-incubated at37° C. for at least five minutes.

The reaction is started by mixing 15 μl of substrate into each ribozymetube by rapid pipetting (note that final ribozyme concentrations are 10,8, 6, 4, 2 nM). 5 μl aliquots are removed at 15 or 30 second intervalsand quenched with 5 μl stop solution (95% formamide, 20 mM EDTA xylenecyanol, and bromphenol blue dyes). Following the final ribozyme timepoint, an aliquot of the remaining substrate is removed as a zeroribozyme control.

The samples are separated on either 15% or 20% polyacrylamide gels. Eachgel is visualized and quantitated with an Ambis beta scanner (AmbisSystems, San Diego, Calif.).

For the most active ribozymes, kinetic analyses are performed insubstrate excess to determine K_(m l and K) _(cat) values.

For kinetic reactions with long RNA substrates (greater than 15 bases inlength) the substrates are prepared by transcription using T7 RNApolymerase and defined templates containing a T7 promoter, and DNAencoding appropriate nucleotides of the viral RNA. The substratesolution is made 1× in assay buffer (75 mM Tris-HCl, pH 7.6; 0.1 mMEDTA; 10 mM MgCl₂) and contains 58 nanomolar concentration of the longRNA molecules. The reaction is started by addition of gel purifiedribozymes to 1 μM concentration. Aliquots are removed at 20, 40, 60, 80and 100 minutes, then quenched by the addition of 5 μl stop solution.Cleavage products are separated using denaturing PAGE. The bands arevisualized and quantitated with an Ambis beta scanner. In one example, along substrate RNA transcript corresponding to nucleotides 1-493 of theHSV-1 ICP4 mRNA is synthesized in vitro with T7 RNA polymerase from adefined template containing T7 promoter and DNA encoding nucleotides1-493 of ICP4.

EXAMPLE 1 Cleavage of Short Substrate RNAs Corresponding to ICP4 GeneTargets

Substrate/ribozyme pairs were evaluated for predicted structuralcharacteristics as described above. Nine candidate substrate/ribozymepairs were tested for their capacity to cleave substrate RNAs in vitroin a ribozyme cleavage assay described above. Of these nine ribozymes,seven were targeted to the 5′ end of the ICP4 mRNA and two ribozymeswere targeted to the 3′ end of the mRNA. Values for the k_(cat), k_(m),and k_(cat)/k_(m) (the “cleavage constants”) were experimentallydetermined for each of these ribozymes based upon the assay reactions.The results are presented in Table II. The k_(cat)/k_(m) values forseveral of these ribozymes are higher than those recently observed forHIV-specific ribozymes in our laboratory. Ribozyme G was extremelyactive in this assay, with a calculated k_(cat)/k_(m) of approximately1'10⁸ M⁻¹ min⁻¹, the highest level of activity observed using thisassay. The generally high level of activity observed suggests thatfactors other than correct folding and the GUC cleavage region influencethe structural characteristics of ribozyme-substrate interaction.

TABLE II RIBOZYME CLEAVAGE OF SHORT ICP4 TARGET RNA SUBSTRATES ICP4Cleavage Ribozyme Site⁻¹ k_(cat)/k_(a) k_(cat) k_(a) A  66 (1) 2 × 10⁷M⁻¹ min⁻¹ B 177 no activity C 200 6 × 10⁷ M⁻¹ min⁻¹ 1.73 min⁻¹ 34 nM D231 no activity E 309 (8) 5.3 × 10⁷ M⁻¹ min⁻¹ F 864,888 3.4 × 10^(6 M−1)min⁻¹ G 870,894 1.17 × 10^(8 M−1) min⁻¹ 5.7 min⁻¹ 127 nM  H 3271  noactivity I 3559  5 × 10⁷ 2.2 min⁻¹ 69 nM ¹Nucleotide number relative toEMBL HSV-1 ICP4 gene sequence. Numbers in parentheses correspond totarget SEQ ID NOS.

EXAMPLE 2 Cleavage of Long Substrate RNA's Corresponding to ICP4 Gene

The ability of three ribozymes to catalyze the cleavage of anapproximately 490 nucleotide long ICP4 RNA substrate (corresponding tonucleotides 1-493 of ICP4 and prepared as described above was determinedusing the ribozyme cleavage assay described above. The assays werepreformed in ribozyme excess and approximate k_(cat)/k_(m) values werecalculated. The results are presented in Table III. The cleavageconstants for short substrate cleavage by each ribozyme are alsoincluded for comparison.

The results indicate that, generally, ribozyme cleavage of longsubstrates proceeds at a slower rate compared to cleavage activities onshort substrates, although cleavage activity is not altogethereliminated by the use of the longer substrate. The effect of using alonger substrate varied among the five ribozymes tested, indicating thatthe accessibility and structural parameters of different cleavage siteschange unpredictably when presented in the context of the longer RNAsubstrates.

All three of the ribozymes exhibited a low capacity to catalyze the longsubstrate relative to their activities on the shorter substrates. Two ofthe ribozymes, ribozymes A and E, were relatively more effective atcatalyzing cleavage of the longer substrate RNA.

These results demonstrate that structural characteristics of thesubstrate RNA may influence ribozyme catalytic activity.

TABLE III EFFECT OF SUBSTRATE RNA SIZE ON RIBOZYME ACTIVITY % ActivitySubstrate K_(cat)/k_(m) on Short Ribozyme Length¹ (M⁻¹ min⁻¹⁾ SubstrateA 17 2 × 10⁷ 100 490 4 × 10⁴ 57 C 17 6 × 10⁷ 100 490 5 × 10² 8 E 17 5.3× 10⁷   100 490 2 × 10³ 17 ¹In nucleotides.

Kinetic Analysis

A simple reaction mechanism for ribozyme-mediated cleavage is:

where R=ribozyme, S=substrate, and P=products. The boxed step isimportant only in substrate excess. Because ribozyme concentration is inexcess over substrate concentration, the concentration of theribozyme-substrate complex ([R:S]) is constant over time except duringthe very brief time when the complex is being initially formed, i.e.,:$\frac{\left\lbrack {R:S} \right\rbrack}{t} = 0$

where t=time, and thus:

(R)(S)k ₁=(RS)(k ₂ +k ₁).

The rate of the reaction is the rate of disappearance of substrate withtime: ${Rate} = {\frac{- {(S)}}{t} = {k_{2}({RS})}}$

Substituting these expressions:${(R)(S)k_{1}} = {{1/k_{2}}\frac{- {(S)}}{t}\left( {k_{2} + k_{1}} \right)\quad \text{or:}}$$\frac{- {(S)}}{s} = {\frac{k_{1}k_{2}}{\left( {k_{2} + k_{1}} \right)}(R){t}}$

Integrating this expression with respect to time yields:${{- \ln}\frac{S}{S_{0}}} = {\frac{k_{1}k_{2}}{\left( {k_{2} + K_{1}} \right)}(R)t}$

where S₀=initial substrate. Therefore, a plot of the negative log offraction substrate uncut versus time (in minutes) yields a straight linewith slope:${slope} = {{\frac{k_{1}k_{2}}{\left( {k_{2} + k_{1}} \right)}(R)} = k_{obs}}$

where k_(obs)=observed rate constant. A plot of slope (k_(obs)) versusribozyme concentration yields a straight line with a slope which is:${{slope} = {\frac{k_{1}k_{2}}{\left( {k_{2} + k_{1}} \right)}\quad {which}\quad {is}\quad \frac{k_{cat}}{K_{m}}}}\quad$

Using these equations the data obtained from the kinetic experimentsprovides the necessary information to determine which ribozyme tested ismost useful, or active. Such ribozymes can be selected and tested in invivo or ex vivo systems.

Liposome Preparation

There follows an example of the entrapment of ribozyme molecules withina liposome drug delivery vehicle. Lipid molecules were dissolved avolatile organic solvent (CHCl₃, methanol, diethylether, ethanol, etc.).The organic solvent was removed by evaporation. The lipid was hydratedinto suspension with 0.1× phosphate buffered saline (PBS), thenfreeze-thawed 3× using liquid nitrogen and incubation at roomtemperature. The suspension was extruded sequentially through a 0.4 μm,0.2 μm and 0.1 μm polycarbonate filters at maximum pressure of 800 psi.The ribozyme was mixed with the extruded liposome suspension andlyophilized to dryness. The lipid/ribozyme powder was rehydrated withwater to one-tenth the original volume. The suspension was diluted tothe minimum volume required for extrusion (0.4 ml for 1.5 ml barrel and1.5 ml for 10 ml barrel) with 1×PBS and re-extruded through 0.4 μm, 0.2μm, 0.1 μm polycarbonate filters. The liposome entrapped ribozyme wasseparated from untrapped ribozyme by gel filtration chromatography(SEPHAROSE CL-4B, BIOGEL A5M). The liposome extractions were pooled andsterilized by filtration through a 0.2 μm filter. The free ribozyme waspooled and recovered by ethanol precipitation. The liposomeconcentration was determined by incorporation of a radioactive lipid.The ribozyme concentration was determined by labeling with ³²P. Rossi etal., 1992, supra (and references cited therein), describe other methodssuitable for preparation of liposomes.

In experiments with a liposome formulation composed of a synthetic lipidderivative disteraoylphosphatidylethylamidothioacetyl succinimide(DSPE-ATS) coformulated with dipalmitoylphosphatidyl choline andcholesterol we observed uptake of 100 and 200 nm diameter liposomes withsimilar kinetics. The larger particles accommodated a larger number ofentrapped molecules, or larger molecular weight molecules, such as anexpression plasmid. These particles showed a linear relationship betweenthe lipid dose offered and the mean log fluorescence (calcine was usedto follow liposome uptake). No cytotoxicity was observed even with a 200μM dose. These liposomes are particularly useful for delivery to CD4cell populations.

In Vivo Assay

The efficacy of action of a chosen ribozyme may be tested in vivo by useof cell cultures sensitive to HSV, using standard procedures. Forexample, monolayer cultures of HSV-sensitive cells are grown in tissueculture plates or in raft cultures of HSV-permissive cells as describedpreviously. Kopan et al., 105 J. Cell Biol. 427, 1987. Prior totransfection with HSV DNA, cultures are treated for three to 24 hourswith ribozyme-containing liposomes. Cells are then rinsed with phosphatebuffered saline (PBS). After transfection the cells are treated forthree to five days with appropriate liposome preparations and mediumchanges. Viral RNA is harvested using the guanidinium isothiocyanateprocedure and the resultant RNA preparation is subjected to analysisusing the RNAse protection assay.

Alternatively, transformed cell types (e.g., Vero cells) are used toassess the ability of ribozyme preparations to cleave HSV mRNA andreduce the expression of target proteins. Cell monolayers are treatedwith ribozyme containing liposome preparations for between 3 and 96 hrs.For RNA analyses, the cells are lysed in guanidine isothiocyanatesolutions and the RNA are analyzed using the RNAse protection assay. Forprotein quantitation, cell lysates are prepared according to methodsknown to those in the art and proteins are quantified by immunoanalysisusing anti-HSV protein specific antibodies.

Ribonuclease Protection Assay

The accumulation of target mRNA in cells or the cleavage of the RNA byribozymes or RNAseH (in vitro or in vivo) can be quantified using anRNAse protection assay.

In this method, antisense riboprobes are transcribed from template DNAusing T7 RNA polymerase (U.S. Biochemicals) in 20 μl reactionscontaining 1× transcription buffer (supplied by the manufacturer), 0.2mM ATP, GTP and UTP, 1 U/μl pancreatic RNAse inhibitor (BoehringerMannheim Biochemicals) and 200 μCi ³²P-labeled CTP (800 Ci/mmol, NewEngland Nuclear) for 1 hour at 37° C. Template DNA is digested with 1 URNAse-free DNAse I (U.S. Biochemicals, Cleveland, Ohio) at 37° C. for 15minutes and unincorporated nucleotides removed by G-50 SEPHADEX spinchromatography.

In a manner similar to the transcription of antisense probe, the targetRNA can be transcribed in vitro using a suitable DNA template. Thetranscript is purified by standard methods and digested with ribozyme at37° C. according to methods described later.

Alternatively, virus-infected cells are harvested into 1 ml of PBS,transferred to a 1.5 ml EPPENDORF tube, pelleted for 30 seconds at lowspeed in a microcentrifuge, and lysed in 70 μl of hybridization buffer(4 M guanidine isothiocyanate, 0.1% sarcosyl, 25 mM sodium citrate, pH7.5). Cell lysate (45 μl) or defined amounts of in vitro transcript(also in hybridization buffer) is then combined with 5 μl ofhybridization buffer containing 5×10⁵ cpm of each antisense riboprobe in0.5 ml EPPENDORF tubes, overlaid with 25 μl mineral oil, andhybridization accomplished by heating overnight at 55° C. Thehybridization reactions are diluted into 0.5 ml RNAse solution (20 U/mlRNAse A, 2 U/ml RNAse T1, 10 U/ml RNAse-free DNAse I in 0.4 M NaCl),heated for 30 minutes at 37° C., and 10 μl of 20% SDS and 10 μl ofProteinase K (10 mg/ml) added, followed by an additional 30 minutesincubation at 37° C. Hybrids are partially purified by extraction with0.5 ml of a 1:1 mixture of phenol/chloroform; aqueous phases arecombined with 0.5 ml isopropanol, and RNAse-resistant hybrids pelletedfor 4.0 minutes at room temperature (about 20° C.) in a microcentrifuge.Pellets are dissolved in 10 μl loading buffer (95% formamide, 1× TBE,0.1% bromophenol blue, 0.1% xylene cylanol), heated to 95° C. for fiveminutes, cooled on ice, and analyzed on 4% polyacrylamide/7 M urea gelsunder denaturing conditions.

Ribozyme Stability

The chosen ribozyme can be tested to determine its stability, and thusits potential utility. Such a test can also be used to determine theeffect of various chemical modifications (e.g., addition of a poly(A)tail) on the ribozyme stability and thus aid selection of a more stableribozyme. For example, a reaction mixture contains 1 to 5 pmoles of 5′(kinased) and/or 31 labeled ribozyme, 15 μg of cytosolic extract and 2.5mM MgCl₂ in a total volume of 100 μl. The reaction is incubated at 37°C. Eight μl aliquots are taken at timed intervals and mixed with 8 μl ofa stop mix (20 mM EDTA, 95% formamide). Samples are separated on a 15%acrylamide sequencing gel, exposed to film, and scanned with an Ambis.

A 3′-labeled ribozyme can be formed by incorporation of the ³²P-labeledcordycepin at the 3′ OH using poly(A) polymerase. For example, thepoly(A) polymerase reaction contains 40 mM Tris, pH 8, 10 mM MgCl₂, 250mM NaCl, 2.5 mM MnCl₂; 3 μl P³² cordycepin, 500 Ci/mM; and 6 unitspoly(A) polymerase in a total volume of 50 μl. The reaction mixture wasincubated for 30 minutes at 37° C.

Effect of Base Substitution upon Ribozyme Activity p To determine whichprimary structural characteristics could change ribozyme cleavage ofsubstrate, minor base changes can be made in the substrate cleavageregion recognized by a specific ribozyme. For example, the substratesequences can be changed at the central “C” nucleotide, changing thecleavage site from a GUC to a GUA motif. The K_(cat)/K_(m) values forcleavage using each substrate are then analyzed to determine if such achange increases ribozyme cleavage rates. Similar experiments can beperformed to address the effects of changing bases complementary to theribozyme binding arms. Changes predicted to maintain strong binding tothe complementary substrate are preferred. Minor changes in nucleotidecontent can alter ribozyme/substrate interactions in ways which areunpredictable based upon binding strength alone. Structures in thecatalytic core region of the ribozyme recognize trivial changes ineither substrate structure or the three dimensional structure of theribozyme/substrate complex.

To begin optimizing ribozyme design, the cleavage rates of ribozymescontaining varied arm lengths, but targeted to the same length of shortRNA substrate can be tested. Minimal arm lengths are required andeffective cleavage varies with ribozyme/substrate combinations.

The cleavage activity of selected ribozymes can be assessed usingHSV-homologous substrates. The assays are performed in ribozyme excessand approximate K_(cat)/K_(min) values obtained. Comparison of valuesobtained with short and long substrates indicates utility in vivo of aribozyme.

Intracellular Stability of Liposome-delivered Ribozymes

To test the stability of a chosen ribozyme in vivo the following test isuseful. Ribozymes are ³²P-end labeled, entrapped in liposomes anddelivered to HSV sensitive cells for three hours. The cells arefractionated and purified by phenol/chloroform extraction. Cells (1×10⁷,T-175 flask) are scraped from the surface of the flask and washed twicewith cold PBS. The cells are homogenized by douncing 35 times in 4 ml ofTSE (10 mM Tris, pH 7.4, 0.25 M Sucrose, mM EDTA). Nuclei are pelletedat 100×g for 10 minutes. Subcellular organelles (the membrane fraction)are pelleted at 200,000×g for two hours using an SW60 rotor. The pelletis resuspended in 1 ml of H buffer (0.25 M Sucrose, 50 mM HEPES, pH7.4). The supernatant contains the cytoplasmic fraction (inapproximately 3.7 ml). The nuclear pellet is resuspended in 1 ml of 65%sucrose in TM (50 mM Tris, pH 74., 2.5 mM MgCl₂) and banded on a sucrosestep gradient (1 ml nuclei in 65% sucrose TM, 1 ml 60% sucrose TM, 1 ml55% sucrose TM, 50% sucrose TM, 300 ul 25% sucrose TM) for one hour at37,000×g with an SW60 rotor. The nuclear band is harvested and dilutedto 10% sucrose with TM buffer. Nuclei are pelleted at 37,000×g using anSW60 rotor for 15 minutes and the pellet resuspended in 1 ml of TMbuffer. Aliquots are size fractionated on denaturing polyacrylamide gelsand the intracellular localization determined. By comparison to themigration rate of newly synthesized ribozyme, the various fractioncontaining intact ribozyme can be determined.

To investigate modifications which would lengthen the half-life ofribozyme molecules intracellularly, the cells may be fractioned as aboveand the purity of each fraction assessed by assaying enzyme activityknown to exist in that fraction.

The various cell fractions are frozen at −70° C. and used to determinerelative nuclease resistances of modified ribozyme molecules. Ribozymemolecules may be synthesized with 5 phosphorothioate (ps), or2′-O-methyl (2′-OMe) modifications at each end of the molecule. Thesemolecules and a phosphodiester version of the ribozyme are end-labeledwith ³²P and ATP using T4 polynucleotide kinase. Equal concentrationsare added to the cell cytoplasmic extracts and aliquots of each taken at10 minute intervals. The samples are size fractionated by denaturingPAGE and relative rates of nuclease resistance analyzed by scanning thegel with an Ambis β-scanner. The results show whether the ribozymes aredigested by the cytoplasmic extract, and which versions are relativelymore nuclease resistant. Modified ribozymes generally maintain 80-90% ofthe catalytic activity of the native ribozyme when short RNA substratesare employed.

Unlabeled, 5′ end-labeled or 3′ end-labeled ribozymes can be used in theassays. These experiments can also be performed with human cell extractsto verify the observations.

EXAMPLE 3 Delivery of Stable Ribozyme Into Vero Cells

Ribozymes were end-labeled, encapsulated in liposomes and delivered toVero cells as described above. Intracellular ribozymes were purifiedfrom cell fractions by phenol/chloroform extraction, and aliquots weresize fractionated along with newly synthesized ribozyme samples ondenaturing polyacrylamide gels to determine intracellular location ofintact ribozymes. By comparison with the migration rate of newlysynthesized ribozymes, only the cytoplasmic fraction was observed tocontain intact ribozyme. Although intact ribozymes recovered from thecytoplasmic fraction represented a relatively small percentage of theribozymes encapsulated into the liposome preparations, these resultsdemonstrate that ribozymes can be delivered to and retain stabilitywithin the cell cytoplasm via a liposome delivery vehicle.

EXAMPLE 4 Synthesis of Modified Ribozymes with Enhanced Resistance toNuclease Digestion

Modified ribozymes were synthesized with 5 phosphorothioate (PS) or 2′O-methyl (2′O—Me) modifications at each end of the molecule, andend-labeled with ³²P-γATP using T4 polynucleotide kinase. Unmodified(phosphodiester) ribosomes were synthesized as described above, andsimilarly end-labeled.

Cultured Vero cells were fractionated as described above. The purity ofeach fraction was assessed by assaying enzyme activities known to residein that fraction. As shown in Table IV, all enzyme activities testedwere found predominantly in the appropriate cell fractions, indicatingan acceptable level of purity. Cell fractions were frozen at −70° C.prior to use in a nuclease resistance assay.

TABLE IV % OF TOTAL ENZYME ACTIVITY Membrane Nuclear Cytoplasmic ENZYMEFraction Fraction Fraction Lactate 3 3 94 Dehydrogenase (cytoplasmicmarker) Calcine 81 3 16 (membrane marker) Lysosome Hexosaminidase 75 520 Glucocerebrosidase 96 0 4 (membrane marker) Endosome Alkaline 97 2 1Phosphodiesterase (membrane marker)

Equal concentrations (1-2 pmoles/100 microliters) of unmodified and PSor 2′ O—Me modified ribozymes were added to Vero cell cytoplasmicextracts and incubated at 37° C. for 0-60 minutes. Aliquots of each weretaken at 10 minute intervals, and size fractionated by electrophoresison denaturing polyacrylamide gels (15%). Relative rates of ribozymedegradation were analyzed by scanning the gels with an Ambis betascanner as measure of relative nuclease resistance.

We found that the unmodified ribozyme is quickly digested by thecytoplasmic extract, but that the PS and 2′ O—Me modified ribozymes arenot digested as rapidly and are thus relatively more nuclease resistant.The PS and 2′ O—Me modified ribozymes retain 80-90% of the catalyticactivity observed with their unmodified ribozyme counterparts, arebetter able to resist nuclease digestion, and therefore may haveincreased half-lifes in vivo. In that connection, PS and 2′ O—Memodified ribozymes may have advantages over unmodified ribozymes for usein therapeutic applications.

In addition, we found that nearly equivalent nuclease resistance assayresults were obtained for unlabeled, 5′ end-labeled and 3′ end-labeledunmodified ribozymes indicating that the observed loss of radioactivelabel in the PS and 2′ O—Me modified ribozymes was not due todephosphorylation of the 5′ end labels, but instead reflects degradationby cytoplasmic nuclease. This suggests that a significant component ofVero cell nuclease activity is endonucleolytic in nature, and it maytherefore be desirable to introduce similar modifications to theinternal regions of the ribozyme molecule in order to best optimizenuclease resistance.

EXAMPLE 5 Ribozyme Cleavage of Single Base-Substituted Substrates InVitro

Unmodified ribozymes I and E, specific for targets within the ICP4 geneof HSV-1, were synthesized and purified as described above.

A single nucleotide change was incorporated into the substrate cleavagesite recognized by ribozyme I. Specifically, the substrate sequenceCCGGGGGUCUUCGCGCG was changed at the central C nucleotide to becomeCCGGGGGUAUUCGCGCG, so that the GUC cleavage site became GUA. Thismodified substrate was synthesized and tested for in vitro cleavage byribozyme I using the ribozyme cleavage assay described above.

Single nucleotide changes were incorporated into the regionscomplementary to the ribozyme binding arms in the substrate RNAsrecognized by ribozymes I and E as indicated in Table V, infra. Computeranalysis of ribozyme/substrate folding predicted that these changeswould result in the maintenance of ribozyme/substrate binding strength.Four such modified substrates were synthesized and tested alongsidetheir “native” substrate counterparts for in vitro cleavage by ribozymesI or E.

Ribozyme I was tested for its ability to cleave two substrate sequencesdiffering by a single base in the substrate cleavage site, one substratecorresponding to nucleotides 3550-3566 of the ICP4 mRNA sequence, andthe other corresponding to the same sequence with a single basesubstitution in the cleavage site. The k_(cat)/k_(m) values for cleavageof both substrates are 5.3×10⁷ M⁻¹ min⁻¹ and 7.3×10⁷ M⁻¹ min⁻¹ for thenative and modified site respectively. The results indicate thatribozyme I is significantly more efficient at cleaving the modifiedsubstrate containing the GUA site. Thus. ribozyme I is not only capableof recognizing and cleaving the “native” ICP4 mRNA target, but is alsocapable of recognizing and cleaving (more efficiently) that same ICP4mRNA target with a point mutation in the cleavage site. Naturalmutations to particular cleavage sites in viral RNA within infected hostcells are possible. Ribozyme I, therefore, may be a particularly goodtherapeutic ribozyme given its target recognition versatility. Theresults also suggest that ICP4 mRNA targets containing a GUA cleavagesite would be acceptable if not preferred ribozyme substrates.

Ribozymes I and E were also tested for their ability to cleavesubstrates with single nucleotide modifications in the complementaryregions. The results, shown in Table V, indicate that both ribozymes canrecognize and efficiently cleave substrates with nucleotide pointmutations. The results also show that relatively minor changes insubstrate sequence can influence ribozyme/substrate interactions in waysthat were not predicted based on binding strength alone.

TABLE V EFFECT OF SUBSTRATE COMPLEMENTARY REGION MODIFICATIONS ONRIBOZYME CATALYTIC ACTIVITY PREDICTED BINDING SUBSTRATE ENERGYk_(cat)/k_(m) RIBOZYME SEQUENCE (kcal/mole) M⁻¹ min⁻¹ ICCGGGGGUCUUCGCGCG −13.3 5.3 × 10⁷ CCUGGGGUCUUCGCGCG −10.9 3.3 × 10⁷CCGGAGGUCUUCGCGCG −5.3 7.5 × 10⁶ E GAUGGCGUCGGAGAACA −11.8   3 × 10⁷GAUGGCGUCGXXAUCA −11.2 3.2 × 10⁷ GAUGGCGUCGGAAAACA −8.0 2.9 × 10⁷ Singlebase changes are indicated in boldface.

EXAMPLE 6 Effect of Ribozyme Arm Length on Cleavage Activity

Variations of ribozymes E and I having longer binding arm lengths weredesigned, synthesized and tested together with their prototype ribozymesfor cleavage of the substrate recognized by the prototypes using aribozyme cleavage assay.

The results are summarized in Table VI, and indicate that the minimalbinding arm lengths required for effective cleavage vary from oneribozyme to another. This indicates that each chosen ribozyme will needto be optimized in its structure on an individual basis.

TABLE VI EFFECT OF RIBOZYME BINDING STEM LENGTH UPON SUBSTRATE CLEAVAGEHybridizing Stem Length Binding (stem I energy Ribozyme stem III)(kcal/mole) k_(cat)/k_(m) E-32 5,5 −9.1 3.8 × 10⁷ M⁻¹ min⁻¹ E-33-65 6,5−9.7 2.8 × 10⁷ M⁻¹ min⁻¹ E-33-56 5,6 −11.2 [NA] E-34 6,6 −11.8   3 × 10⁷M⁻¹ min⁻¹ I-32 5,5 −9.4 1.6 × 10⁷ M⁻¹ min⁻¹ I-33-65 6,5 −10.1 <<1 × 10⁶M⁻¹ min⁻¹  I-33-56 5,6 −12.8   1 × 10⁶ M⁻¹ min⁻¹ I-34 6,6 −13.3   5 ×10⁷ M⁻¹ min⁻¹ NA = not available.

EXAMPLE 7

We have used the ribonuclease protection assay to determine thelocalization of the HSV ICP4 and ICP27 mRNAs within infected Vero cells.We found that>80% of the viral mRNAs were situated in the cytoplasm ofthe infected cells at 4 hours post infection. We have chosen a sitewithin the HSV ICP4 mRNA which is accessible in RNAse H assays forfurther study. We have examined the effects of altering ribozyme bindingarm lengths on catalytic activity and chosen a ribozyme which hasbinding arms of 6 nucleotides. We call this molecule RPI 1197. (See,Draper, “Constructs for High Yield Ribozyme Production”, filed on thesame day as the present application, hereby incorporated by referenceherein.) We have checked the ability of RPI 1197 to cleave the ICP4 mRNAin cell lysates from infected cells and found that the amount of ICP4cleaved by added RPI 1197 increased over time and gave the anticipatedcleavage products. Using a liposome formulation which delivers ribozymesto the cytoplasm of cells, we pre-treated cells with two ribozymemolecules (RPI 1197 and RPI 1200 [a 2′-O-Methyl substituted version of1197]) and tested them for their ability to reduce intracellular levelsof the viral ICP4 mRNA and inhibit viral replication. We found that RPI1197 and RPI 1200 reduced the mRNA levels by 10 and 36%, respectively,and viral load by 6 and 18%, respectively.

Early testing of the accessible sites of the UL5 transcript using theRNAse H assay has demonstrated that a number of regions within thetranscript are accessible to binding of ribozymes. We have used a 622nucleotide region (region D) of the UL5 RNA to check the cleavageability of ribozymes targeted to both weakly and strongly accessiblesites. We have found that the ribozyme activity in this fragment of RNAshowed a good correlation between accessibility of sites and the abilityof ribozymes to cleave the target RNA at those sites. In preliminaryexperiments, we have observed that the results of the RNAse H assay canalso be mimicked using a gel-binding assay in which pieces ofcomplementary nucleic acid (either RNA or DNA) are bound to target RNAand the mixtures are electrophoresed through gels to observe theformation of oligo/target complexes. By varying the concentration of theoligonucleotide, one can determine the accessible regions of the RNA aswell as the binding affinity of the oligonucleotide for that region. Wehave found a good correlation between the strength of the bindingcomplexes at particular sites and the activity of ribozymes at thosesites within the UL5 RNA molecules.

Administration of Ribozyme

Selected ribozymes can be administered prophylactically, or to virusinfected patients, e.g., by exogenous delivery of the ribozyme to aninfected tissue by means of an appropriate delivery vehicle, e.g., aliposome, a controlled release vehicle, by use of iontophoresis,electroporation or ion paired molecules, or covalently attached adducts,and other pharmacologically approved methods of delivery. Routes ofadministration include intramuscular, aerosol, oral (tablet or pillform), topical, systemic, ocular, intraperitoneal and/or intrathecal.Expression vectors for immunization with ribozymes and/or delivery ofribozymes are also suitable.

The specific delivery route of any selected ribozyme will depend on theuse of the ribozyme. Generally, a specific delivery program for eachribozyme will focus on naked ribozyme uptake with regard tointracellular localization, followed by demonstration of efficacy.Alternatively, delivery to these same cells in an organ or tissue of ananimal can be pursued. Uptake studies will include uptake assays toevaluate cellular oligonucleotide uptake, regardless of the deliveryvehicle or strategy. Such assays will also determine the intracellularlocalization of the oligonucleotide following uptake, ultimatelyestablishing the requirements for maintenance of steady-stateconcentrations within the cellular compartment containing the targetsequence (nucleus and/or cytoplasm). Efficacy and cytotoxicity can thenbe tested. Toxicity will not only include cell viability but also cellfunction.

Some methods of delivery that may be used include:

a. encapsulation in liposomes,

b. transduction by retroviral vectors,

c. conjugation with cholesterol,

d. localization to nuclear compartment utilizing antigen binding sitefound on most snRNAs,

e. neutralization of charge of ribozyme by using nucleotide derivatives,and

f. use of blood stem cells to distribute ribozymes throughout the body.

At least three types of delivery strategies are useful in the presentinvention, including: ribozyme modifications, particle carrier drugdelivery vehicles, and retroviral expression vectors. Unmodifiedribozymes and antisense oligonucleotides, like most small molecules, aretaken up by cells, albeit slowly. To enhance cellular uptake, theribozyme may be modified essentially at random, in ways which reducesits charge but maintains specific functional groups. This results in amolecule which is able to diffuse across the cell membrane, thusremoving the permeability barrier.

Modification of ribozymes to reduce charge is just one approach toenhance the cellular uptake of these larger molecules. The randomapproach, however, is not advisable since ribozymes-are structurally andfunctionally more complex than small drug molecules. The structuralrequirements necessary to maintain ribozyme catalytic activity are wellunderstood by those in the art. These requirements are taken intoconsideration when designing modifications to enhance cellular delivery.The modifications are also designed to reduce susceptibility to nucleasedegradation. Both of these characteristics should greatly improve theefficacy of the ribozyme. Cellular uptake can be increased by severalorders of magnitude without having to alter the phosphodiester linkagesnecessary for ribozyme cleavage activity.

Chemical modifications of the phosphate backbone will reduce thenegative charge allowing free diffusion across the membrane. Thisprinciple has been successfully demonstrated for antisense DNAtechnology. The similarities in chemical composition between DNA and RNAmake this a feasible approach. In the body, maintenance of an externalconcentration will be necessary to drive the diffusion of the modifiedribozyme into the cells of the tissue. Administration routes which allowthe diseased tissue to be exposed to a transient high concentration ofthe drug, which is slowly dissipated by systemic adsorption arepreferred. Intravenous administration with a drug carrier designed toincrease the circulation half-life of the ribozyme can be used. The sizeand composition of the drug carrier restricts rapid clearance from theblood stream. The carrier, made to accumulate at the site of infection,can protect the ribozyme from degradative processes.

Drug delivery vehicles are effective for both systemic and topicaladministration. They can be designed to serve as a slow releasereservoir, or to deliver their contents directly to the target cell. Anadvantage of using direct delivery drug vehicles is that multiplemolecules are delivered per uptake. Such vehicles have been shown toincrease the circulation half-life of drugs which would otherwise berapidly cleared from the blood stream. Some examples of such specializeddrug delivery vehicles which fall into this category are liposomes,hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesivemicrospheres.

From this category of delivery systems, liposomes are preferred.Liposomes increase intracellular stability, increase uptake efficiencyand improve biological activity.

Liposomes are hollow spherical vesicles composed of lipids arranged in asimilar fashion as those lipids which make up the cell membrane. Theyhave an internal aqueous space for entrapping water soluble compoundsand range in size from 0.05 to several microns in diameter. Severalstudies have shown that liposomes can deliver RNA to cells and that theRNA remains biologically active.

For example, a liposome delivery vehicle originally designed as aresearch tool, Lipofectin, has been shown to deliver intact mRNAmolecules to cells yielding production of the corresponding protein.

Liposomes offer several advantages: They are non-toxic and biodegradablein composition; they display long circulation half-lives; andrecognition molecules can be readily attached to their surface fortargeting to tissues. Finally, cost effective manufacture ofliposome-based pharmaceuticals, either in a liquid suspension orlyophilized product, has demonstrated the viability of this technologyas an acceptable drug delivery system.

Other controlled release drug delivery systems, such as nonoparticlesand hydrogels may be potential delivery vehicles for a ribozyme. Thesecarriers have been developed for chemotherapeutic agents andprotein-based pharmaceuticals, and consequently, can be adapted forribozyme delivery.

Topical administration of ribozymes is advantageous since it allowslocalized concentration at the site of administration with minimalsystemic adsorption. This simplifies the delivery strategy of theribozyme to the disease site and reduces the extent of toxicologicalcharacterization. Furthermore, the amount of material to be applied isfar less than that required for other administration routes. Effectivedelivery requires the ribozyme to diffuse into the infected cells.Chemical modification of the ribozyme to neutralize negative charge maybe all that is required for penetration. However, in the event thatcharge neutralization is insufficient, the modified ribozyme can beco-formulated with permeability enhancers, such as Azone or oleic acid,in a liposome. The liposomes can either represent a slow releasepresentation vehicle in which the modified ribozyme and permeabilityenhancer transfer from the liposome into the infected cell, or theliposome phospholipids can participate directly with the modifiedribozyme and permeability enhancer in facilitating cellular delivery. Insome cases, both the ribozyme and permeability enhancer can beformulated into a suppository formulation for slow release.

Ribozymes may also be systemically administered. Systemic absorptionrefers to the accumulation of drugs in the blood stream followed bydistribution throughout the entire body. Administration routes whichlead to systemic absorption include: intravenous, subcutaneous,intraperitoneal, intranasal, intrathecal and ophthalmic. Each of theseadministration routes expose the ribozyme to an accessible diseasedtissue. Subcutaneous administration drains into a localized lymph nodewhich proceeds through the lymphatic network into the circulation. Therate of entry into the circulation has been shown to be a function ofmolecular weight or size. The use of a liposome or other drug carrierlocalizes the ribozyme at the lymph node. The ribozyme can be modifiedto diffuse into the cell, or the liposome can directly participate inthe delivery-of either the unmodified or modified ribozyme to the cell.

A liposome formulation which can associate ribozymes with the surface oflymphocytes and macrophages is also useful. This will provide enhanceddelivery to HSV-infected cells by taking advantage of the specificity ofmacrophage and lymphocyte immune recognition of infected cells. Wholeblood studies show that the formulation is taken up by 90% of thelymphocytes after 8 hours at 37° C. Preliminary biodistribution andpharmacokinetic studies yielded 70% of the injected dose/gm of tissue inthe spleen after one hour following intravenous administration.

Intraperitoneal administration also leads to entry into the circulationwith the molecular weight or size of the ribozyme-delivery vehiclecomplex controlling the rate of entry.

Liposomes injected intravenously show accumulation in the liver, lungand spleen. The composition and size can be adjusted so that thisaccumulation represents 30% to 40% of the injected dose. The rest isleft to circulate in the blood stream for up to 24 hours.

The chosen method of delivery will result in cytoplasmic accumulation inthe afflicted cells and molecules should have some nuclease-resistancefor optimal dosing. Nuclear delivery may be used but is less preferable.Most preferred delivery methods include liposomes (10-400 nm),hydrogels, controlled-release polymers, microinjection orelectroporation (for ex vivo treatments) and other pharmaceuticallyapplicable vehicles. The dosage will depend upon the disease indicationand the route of administration but should be between 100-200 mg/kg ofbody weight/day. The duration of treatment will extend through thecourse of the disease symptoms, usually at least 14-16 days and possiblycontinuously. Multiple daily doses are anticipated for topicalapplications, ocular applications and vaginal applications. The numberof doses will depend upon disease delivery vehicle and efficacy datafrom clinical trials.

Establishment of therapeutic levels of ribozyme within the cell isdependent upon the rate of uptake and degradation. Decreasing the degreeof degradation will prolong the intracellular half-life of the ribozyme.Thus, chemically modified ribozymes, e.g., with modification of thephosphate backbone, or capping of the 5′ and 3′ ends of the ribozymewith nucleotide analogs may require different dosaging. Descriptions ofuseful systems are provided in the art cited above, all of which ishereby incorporated by reference herein.

Other embodiments are within the following claims.

SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 115(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 1: AGAGACAGAC CGUCAGACGC UC22 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 15 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CCGGGACGCC GAUAC 15 (2)INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GGAUCGGCCG UCCCUGUCCU 20 (2)INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 4: ACCCAAGCAU CGACCGGUCC 20 (2)INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:19 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GGUCUCGCCC CCUCCCCCC 19 (2)INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:31 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 6: UAGGUGACCU ACCGUGCUACGUCCGCCGUC G 31 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 13 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 7:UAUCCCCGGA GGA 13 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 31 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 8:GGCGUCGGAG AACAAGCAGC GCCCCGGCUC C 31 (2) INFORMATION FOR SEQ ID NO: 9:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQID NO: 9: CACGACCUCG AC 12 (2) INFORMATION FOR SEQ ID NO: 10: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQID NO: 10: CGCCGUCUCG CCGCGACAGC UGGCUCUGCU G 31 (2) INFORMATION FOR SEQID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCEDESCRIPTION: SEQ ID NO: 11: GUCCGGACGA UCCCGACGCC C 21 (2) INFORMATIONFOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)SEQUENCE DESCRIPTION: SEQ ID NO: 12: GACGACGAUG ACGGGGACGA GUACGACGAC G31 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 15 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GUAUCCGGAC CCCAC 15 (2)INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:13 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 14: CGUCGUCACG GCC 13 (2)INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:9 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 15: CAUAGACCT 9 (2) INFORMATIONFOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)SEQUENCE DESCRIPTION: SEQ ID NO: 16: GUCCGCAUCC UCU 13 (2) INFORMATIONFOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)SEQUENCE DESCRIPTION: SEQ ID NO: 17: GCAUCGAGCG CC 12 (2) INFORMATIONFOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)SEQUENCE DESCRIPTION: SEQ ID NO: 18: GGGCCGCUUC ACGGCCGGGC AG 22 (2)INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:18 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 19: GCGACGCCGG UUCGAGGC 18 (2)INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:14 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 20: ACGCCCUGAU CACG 14 (2)INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:17 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 21: GGGGUGGCUC CAGAACC 17 (2)INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:31 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 22: AACAGCAGCU CCUUCAUCACCGGCAGCGUG G 31 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 13 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 23:GCCUGGCGCA CGC 13 (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 12 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 24:CCGUGGCCAU GA 12 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 12 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 25:AUACGACCGC GC 12 (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 23 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 26:GCGCAGAAGG GCUUCCUGCU GAC 23 (2) INFORMATION FOR SEQ ID NO: 27: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQID NO: 27: GCCUGCCGCG GGAUCCUGGA GGCGCUGG 28 (2) INFORMATION FOR SEQ IDNO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCEDESCRIPTION: SEQ ID NO: 28: CCUGCUGUUU GACAACCAGA GCCUGC 26 (2)INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:15 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 29: AAGCGCAAGA GUCCC 15 (2)INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:17 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 30: GCCCCCCUCC CCGCGCC 17 (2)INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:12 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 31: CCUCCACGCC CC 12 (2)INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:19 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 32: GCGCCCCGUG GCCGUGUCG 19 (2)INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 33: CCUGGAGGCC UACUGCUCCC CG 22(2) INFORMATION FOR SEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 34: CUGUUCCCCG UCCCCUGGCGAC 22 (2) INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 13 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 35: UCAUGUUUGACCC 13 (2) INFORMATION FOR SEQ ID NO: 36: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 26 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 36: UGGCCUCGAUCGCCGCGCGG UGCGCC 26 (2) INFORMATION FOR SEQ ID NO: 37: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 23 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 37:GACGACGACG AUAACCCCCA CCC 23 (2) INFORMATION FOR SEQ ID NO: 38: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQID NO: 38: AUCCCCGACC CCGAGGACGU GCGC 24 (2) INFORMATION FOR SEQ ID NO:39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 (B) TYPE: nucleic acid(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION:SEQ ID NO: 39: CCCGACGUGU CG 12 (2) INFORMATION FOR SEQ ID NO: 40: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQID NO: 40: GUGCUGGCGG CGGCGGGGGC CGUGGA 26 (2) INFORMATION FOR SEQ IDNO: 41: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCEDESCRIPTION: SEQ ID NO: 41: GGAGGCGGGC UUGGCCAC 18 (2) INFORMATION FORSEQ ID NO: 42: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCEDESCRIPTION: SEQ ID NO: 42: CUGGGACGAA GAC 13 (2) INFORMATION FOR SEQ IDNO: 43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCEDESCRIPTION: SEQ ID NO: 43: GGGUGCUGUA ACGG 14 ***** (2) INFORMATION FORSEQ ID NO: 44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCEDESCRIPTION: SEQ ID NO: 44: GUGAACCUUU ACCCAGCCGU CCUC 24 (2)INFORMATION FOR SEQ ID NO: 45: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:16 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 45: GCACAGCGCU UCCGUG 16 (2)INFORMATION FOR SEQ ID NO: 46: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:23 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 46: AGCGCCAGCU AGACGGACAG AAA 23(2) INFORMATION FOR SEQ ID NO: 47: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 18 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 47: CACCUUCAGC AACCCGGG 18(2) INFORMATION FOR SEQ ID NO: 48: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 13 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 48: UAAGCGCAUC CGA 13 (2)INFORMATION FOR SEQ ID NO: 49: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:11 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 49: CUCGCAACAA C 11 (2)INFORMATION FOR SEQ ID NO: 50: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:28 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 50: CGCAAGUGCC CCAUCUGCAG UGGUUCCG28 (2) INFORMATION FOR SEQ ID NO: 51: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 20 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 51: GCGGCCUUAG AGUCCCCCGC20 (2) INFORMATION FOR SEQ ID NO: 52: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 21 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 52: GGUGUAUCUU AUCACCGGCA A21 (2) INFORMATION FOR SEQ ID NO: 53: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 15 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 53: GCUCCGGAAA GAGCA 15 (2)INFORMATION FOR SEQ ID NO: 54: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:23 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 54: CAGACAAUCA ACGAGGUCUU GGA 23(2) INFORMATION FOR SEQ ID NO: 55: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 28 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 55: UGGUGACGGG CGCCACGCGCAUUGCGGC 28 (2) INFORMATION FOR SEQ ID NO: 56: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 16 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 56:CCAAAACAUG UACGCC 16 (2) INFORMATION FOR SEQ ID NO: 57: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 57:UCAACACCAU CUUUCAUGAA UU 22 (2) INFORMATION FOR SEQ ID NO: 58: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQID NO: 58: CCAACUGGGA CAGUACCCGU ACACCCUGAC CA 32 (2) INFORMATION FORSEQ ID NO: 59: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCEDESCRIPTION: SEQ ID NO: 59: ACCUGCAGCG ACGAGAUCUG ACGUACUACU GG 32 (2)INFORMATION FOR SEQ ID NO: 60: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:19 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 60: ACGAAGCGCG CCCUGGCCG 19 (2)INFORMATION FOR SEQ ID NO: 61: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:17 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 61: CCUGACGCGG UUGGCCC 17 (2)INFORMATION FOR SEQ ID NO: 62: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:15 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 62: CUUUACCCGC AGCAA 15 (2)INFORMATION FOR SEQ ID NO: 63: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:14 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 63: UCGUCAUCGA CGAG 14 (2)INFORMATION FOR SEQ ID NO: 64: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:25 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 64: GCCGGGCUCC UUGGGCGUCA CCUCC 25(2) INFORMATION FOR SEQ ID NO: 65: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 13 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 65: GCCGUGGUGU AUU 13 (2)INFORMATION FOR SEQ ID NO: 66: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:16 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 66: CGGCCCGCCU GCGGCC 16 (2)INFORMATION FOR SEQ ID NO: 67: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:16 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 67: CCUGGAGUCG ACCUUC 16 (2)INFORMATION FOR SEQ ID NO: 68: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:11 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 68: CGUCCGCCAG A 11 (2)INFORMATION FOR SEQ ID NO: 69: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:32 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 69: UCAUCUGCAA CCGCACGCUGCGCGAGUACG CC 32 (2) INFORMATION FOR SEQ ID NO: 70: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 70:CGCCUCUCGU AUAGCUGGGC CA 22 (2) INFORMATION FOR SEQ ID NO: 71: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQID NO: 71: UUUUUAUUAA CAACAAAC 18 (2) INFORMATION FOR SEQ ID NO: 72: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQID NO: 72: ACCUCAUGAA GGUGCUGGAG UACGGCC 27 (2) INFORMATION FOR SEQ IDNO: 73: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCEDESCRIPTION: SEQ ID NO: 73: GGCCUGCCCA UCACCGAGGA GCACAUGC 28 (2)INFORMATION FOR SEQ ID NO: 74: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 74: CCGGAAAACU ACAUCACCAA CC 22(2) INFORMATION FOR SEQ ID NO: 75: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 21 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 75: CCGCCAACCU CCCCGGCUGG A21 (2) INFORMATION FOR SEQ ID NO: 76: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 30 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 76: UGUUCUCCUC CCACAAAGAGGUGAGCGCGU 30 (2) INFORMATION FOR SEQ ID NO: 77: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 11 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 77:ACCCGUGAGG G 11 (2) INFORMATION FOR SEQ ID NO: 78: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 11 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 78:CUUACGUUCG U 11 (2) INFORMATION FOR SEQ ID NO: 79: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 17 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 79:CAAGGAGUUU GACGAAU 17 (2) INFORMATION FOR SEQ ID NO: 80: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 80:CGGCCUGACG AUUGAAAA 18 (2) INFORMATION FOR SEQ ID NO: 81: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 25 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 81:AUCACCAACU ACUCGCAGAG CCAGG 25 (2) INFORMATION FOR SEQ ID NO: 82: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQID NO: 82: GAGGUGCACA GCAAACA 17 (2) INFORMATION FOR SEQ ID NO: 83: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQID NO: 83: UGGUCGUGGC CCGCAAC 17 (2) INFORMATION FOR SEQ ID NO: 84: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQID NO: 84: CUCAACAGCC AGAUCGCGGU GACCGC 26 (2) INFORMATION FOR SEQ IDNO: 85: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCEDESCRIPTION: SEQ ID NO: 85: GCGCCUGCGA AAACUGGUUU UU 22 (2) INFORMATIONFOR SEQ ID NO: 86: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)SEQUENCE DESCRIPTION: SEQ ID NO: 86: GCUUUGUAAA GACUC 15 (2) INFORMATIONFOR SEQ ID NO: 87: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)SEQUENCE DESCRIPTION: SEQ ID NO: 87: ACAACUUUCU GCAGCGCCCG 20 (2)INFORMATION FOR SEQ ID NO: 88: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:11 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 88: UGCGACCCAG A 11 (2)INFORMATION FOR SEQ ID NO: 89: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:30 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 89: UCGCCUACGC CCGCAUGGGAGAACUAACGG 30 (2) INFORMATION FOR SEQ ID NO: 90: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 15 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 90:UUGAUUUUAA GCAAC 15 (2) INFORMATION FOR SEQ ID NO: 91: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 11 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 91:CCCGGACGAU U 11 (2) INFORMATION FOR SEQ ID NO: 92: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 9 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 92:UGGACGAAC 9 (2) INFORMATION FOR SEQ ID NO: 93: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 32 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 93:AACAGCUCGA CGUGUUUUAC UGCCACUACA CC 32 (2) INFORMATION FOR SEQ ID NO:94: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 (B) TYPE: nucleic acid(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION:SEQ ID NO: 94: CCGCCGUUCA CACCCAGUUU GCGC 24 (2) INFORMATION FOR SEQ IDNO: 95: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCEDESCRIPTION: SEQ ID NO: 95: CGGGCCUUCC UCGGGAGAUU CCGAAU 26 (2)INFORMATION FOR SEQ ID NO: 96: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:13 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 96: AAGAGCUCUU CGG 13 (2)INFORMATION FOR SEQ ID NO: 97: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:16 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 97: CAUUUGAAGU CGCCCC 16 (2)INFORMATION FOR SEQ ID NO: 98: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:30 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 98: CGUACGUGGA CAACGUUAUCUUCCGGGGCU 30 (2) INFORMATION FOR SEQ ID NO: 99: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 11 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 99:AUGCUGACCG G 11 (2) INFORMATION FOR SEQ ID NO: 100: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 23 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 100:CGCGCGGGGG GCUGAUGUCC GUC 23 (2) INFORMATION FOR SEQ ID NO: 101: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQID NO: 101: AGACGGACAA UUAUACGCUC AU 22 (2) INFORMATION FOR SEQ ID NO:102: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 (B) TYPE: nucleic acid(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION:SEQ ID NO: 102: CGCACGGGUG UUU 13 (2) INFORMATION FOR SEQ ID NO: 103:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQID NO: 103: CCAACGUGGC CGAGUUACUG GAAGAGG 27 (2) INFORMATION FOR SEQ IDNO: 104: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCEDESCRIPTION: SEQ ID NO: 104: CCCCCCUGCC U 11 (2) INFORMATION FOR SEQ IDNO: 105: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCEDESCRIPTION: SEQ ID NO: 105: CACGGCUUCA UGUCCGUCGU CAACAC 26 (2)INFORMATION FOR SEQ ID NO: 106: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 15 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 106: CAACACCCAA CAUCA 15(2) INFORMATION FOR SEQ ID NO: 107: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 29 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 107: GCCAUGGCCA UAAACGCCGACUACGGCAU 29 (2) INFORMATION FOR SEQ ID NO: 108: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 26 (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 108:ACAAGGUCGC CAUCUGCUUU ACGCCC 26 (2) INFORMATION FOR SEQ ID NO: 109: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQID NO: 109: GGCAACCUGC GCCUCAAC 18 (2) INFORMATION FOR SEQ ID NO: 110:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQID NO: 110: CCUCCUCCGA AUUCCUUCGC AU 22 (2) INFORMATION FOR SEQ ID NO:111: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 (B) TYPE: nucleic acid(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION:SEQ ID NO: 111: CGAUGACGUC AU 12 (2) INFORMATION FOR SEQ ID NO: 112: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCE DESCRIPTION: SEQID NO: 112: UCGGCUCUGC GCGAUCCGAA CGUGGUCAUU G 31 (2) INFORMATION FORSEQ ID NO: 113: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) SEQUENCEDESCRIPTION: SEQ ID NO: 113: UCUAUUAACC CGCCGUCCCC UUAC 24 (2)INFORMATION FOR SEQ ID NO: 114: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 19 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 114: GGGGGACUCA CUACCCACC19 (2) INFORMATION FOR SEQ ID NO: 115: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 23 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 115: GCGAGAUGUC CAAUCCACAGACG 23

What is claimed is:
 1. An enzymatic RNA molecule which cleaves RNAencoded by a herpes simplex virus (HSV) in the ICP4 gene.
 2. Theenzymatic RNA molecule of claim 1 which cleaves a sequence selected fromthe group consisting of SEQ ID NO: 1-43.
 3. The enzymatic RNA moleculeof claim 2 which cleaves a sequence comprising SEQ ID NO: 1 or SEQ IDNO:
 8. 4. The enzymatic RNA molecule of claim 1, wherein said enzymaticRNA molecule is selected from the group consisting of enzymatic RNAmolecules that cleave the ICP4 gene at residue 66, 200, 309, 864, 888,870, 894 or
 3559. 5. The enzymatic RNA molecule of claim 1, wherein saidRNA molecule is in a hammerhead motif.
 6. The enzymatic RNA molecule ofclaim 1, wherein said RNA molecule is in a hairpin, hepatitis Deltavirus, group I intron, or RNAse P RNA motif.
 7. An isolated mammaliancell comprising an enzymatic molecule of claim
 1. 8. An isolatedmammalian cell comprising an enzymatic molecule of claim
 4. 9. The cellof claim 7 or claim 8, wherein said cell is a human cell.
 10. A vectorcomprising nucleic acid encoding the enzymatic RNA molecule of claim 1.11. A vector comprising nucleic acid encoding the enzymatic RNA moleculeof claim 4.